Abstract

Four new polyketides have been identified in culture filtrates of the fungal strain Penicillium sp. IBWF104-06 isolated from a soil sample. They are structurally based on the same trans-decalinpentanoic acid skeleton as tanzawaic acids A–H. One of the new compounds was found to inhibit the conidial germination in the rice blast fungus Magnaporthe oryzae at concentrations of 25 μg/mL.

Introduction

Nature still represents the richest source of new antimicrobials which can be attractive as lead structures or biochemical tools for target identification for medical applications as well as for crop science. In both areas, the omnipresent development of resistance results in a demand for new active principles for which the structural diversity of nature holds an excellent supply. While natural products, their synthesis, evolution, and ecological role are of academic interest, compounds identified might also be relevant for application. In the course of investigations into the secondary metabolism of fungal species for the identification of novel lead structures for agrochemical and pharmaceutical research, we isolated a fungal strain from a soil sample. The culture filtrate extract of this organism was found to inhibit the conidial germination in the rice blast fungus Magnaporthe oryzae, one of the most devastating plant diseases on cultivated rice. Bioactivity-guided fractionation of organic extracts led to the isolation of a series of eight pure compounds, the structures of which were elucidated by 2D NMR spectroscopy as well as by mass spectrometry.

Results and Discussion

Structure elucidation. Compound 1 has the molecular formula C18H26O4 determined on the basis of NMR and HRMS-ESI data which gave a pseudo-molecular ion at m/z 329.1744 (calcd for [M + Na]+: 329.1729). This composition accounted for six double bond equivalents. The NMR spectra of 1 (Table 1 and Table 2) displayed signals of a (2E,4E)-penta-2,4-dienoic acid moiety including a carbon of a conjugated carbonyl group at δC 170.9 and four olefinic resonances at δH 5.86 (d, J = 15.3 Hz)/δC 122.0, δH 7.28 (dd, J = 11.2, 15.3 Hz)/δC 145.9, δH 6.45 (dd, J = 11.2, 15.2 Hz)/128.5, and δH 6.10 (d, J = 15.2 Hz)/δC 144.8. Moreover, these spectra showed signals of three CH3 groups, two CH2 groups, six CH groups, and two quaternary carbons (Table 1 and Table 2). These data suggested a structural similarity of 1 to the tanzawaic acids, secondary metabolites from Penicillium citrinum[1]. COSY and HMBC correlations (Figure 1) revealed the presence of a trans-decalin scaffold containing six CH groups, two CH2 groups and two quaternary carbons as the hydrogen at δH 1.39 (H-7) showed correlations with hydrogens at δH 1.59 (H-12) and 0.72 (H-9) while the latter correlated with the proton at δH 1.45 (H-10). Likewise, the resonance at δH 1.45 had a correlation with the proton at δH 1.17 (H-11ax) which in turn correlated with those at δH 1.54 (H-11eq) and 1.59 (H-12) forming the western part of the bicyclic framework; further homonuclear interactions were revealed from H-12 (δH 1.59) to H-13 (δH 3.80) and H-14 (δH 5.69). The eastern part of the decalin system was established based on HMBC correlations between the olefinic proton at δH 5.69 (H-14) and the carbons C-12 (δC 41.4), C-13 (δC 67.6), and C-6 (δC 79.0) while the hydrogen of the oxymethine at δH 3.80 (H-13) correlated with the carbons C-7 (δC 49.8), C-14 (δC 127.0) and C-15 (δC 142.9). Based on the COSY and HMBC interactions as well as their 1D multiplicity, the CH3 groups at δH 0.90 (H-17), 1.09 (H-18) and 1.62 (H-16) were attached to the carbons resonating at δC 32.9 (C-10), 35.3 (C-8), and 142.9 (C-15) respectively, while the penta-2,4-dienoic acid unit was connected to the carbon at δC 79.0 (C-6).

The relative configuration of the stereogenic centers was assigned based on NOESY data (Figure 1) and the previous work reported by Malmstrøm et al. on the stereochemistry of two related compounds, tanzawaic acids E and F [2].

The NOESY spectrum showed contacts (Figure 1) between the proton of the oxymethine group at δH 3.80 (H-13) and the protons at δH 1.54 (H-11eq) and 1.59 (H-12) suggesting an axial orientation of the OH group at C-13. The proton at δH 1.17 (H-11ax) correlated with those at δH 0.90 (H-17), 0.72 (H-9ax), and 1.39 (H-8) while H-9ax showed same interactions with the methyl protons at δH 1.09 (H-18) and 0.90 (H-17) suggesting them to be oriented equatorially. The same orientation was suggested for the unsaturated side chain since correlations were found between the proton at δH 6.10 (H-5) and those at 1.09 (H-18) and 1.39 (H-7).

The foregoing data in conjunction with those of the structurally related tanzawaic acid E [2] led to the identification of 1 as a new member of this compound class and it was given the name tanzawaic acid I (Figure 5).

In compound 2, NOESY correlations (Figure 2) observed between H-11ax (δH 1.17) and H-9ax (δH 0.78), H-13 (δH 3.75) and H-7 (δH 1.52) revealed the oxymethine at δH 3.75 (dd, J = 2.7, 6.0 Hz)/δC 68.0 to be responsible for the diastereomery between 1 and 2 due to inversion of the configuration at C-13. Further NOESY correlations were observed between the proton resonating at δH 0.78 (H-9ax) and the methyl protons at δH 0.91 (Me-17 and Me-18) while H-18 showed the same interactions with the olefinic protons at δH 6.16 (H-5) and 6.45 (H-4). This observation suggested 2 to have the same relative configuration as 1 at C-6, C-7, C-8, and C-10. The above-mentioned data allowed to identify compound 2 as another member of the tanzawaic acid family and was given the name tanzawaic acid J.

The molecular formula of compound 3, C18H26O3 was established by NMR in conjunction with HRMS-ESI which gave a pseudo-molecular ion at m/z 313.1788 (calcd for [M + Na]+: 313.1780). This composition required six double bond equivalents and differed from those of compounds 1 and 2 by one oxygen atom. This observation was supported by the absence of oxymethine signals in the NMR spectra. However, features similar to those of the (2E,4E)-penta-2,4-dienoic acid moiety of compounds 1 and 2 and some signal patterns of the trans-decalin part different from those of the two other compounds were observed. The resonance of H-5 (δH 6.31, J = 10.6, 15.0 Hz) was found as a doublet of doublets suggesting the side chain to be attached to a methine carbon in the bicyclic portion. This conclusion was confirmed by a COSY-correlation to H-6 [δH 2.40 (t, J = 10.6 Hz)]. Furthermore, two signals of a cis olefin were unveiled at δH 5.41 (d, J = 10.0 Hz)/δC 132.9 and δH 5.48 (dd, J = 2.4, 10.0 Hz)/δC 135.2 and originated from the protons bound to C-13 and C-14. This supposition was consistent with diagnostic HMBC correlations connecting the protons [δH 1.18 (s)] of the C-16 methyl group to C-6 (δC 58.8), C-15 (δC 74.5) as well as C-14 (δC 135.2) (Figure 3).

The NOESY spectrum of compound 3 revealed similar correlations as in its analogues 1 and 2 between the olefinic proton H-5 (δH 6.31) and the CH3 group attached to C-8 suggesting the same relative orientation of the side chain and the CH3 group (Figure 3) which is supported by a correlation between H-5 and the CH3 (C-16) group (δH 1.18). The complete assignment led to structure 3 as shown in Figure 5. The compound was named tanzawaic acid K.

The molecular formula of compound 4, C18H24O3 was determined based on its NMR and HRMS-ESI data which gave a pseudo-molecular ion at m/z 311.1630 (calcd for [M + Na]+: 311.1623). This composition required seven double bond equivalents. It differed from compound 3 by two hydrogen atoms suggesting one additional carbon–carbon double bond. This assumption was justified by the presence of four olefinic signals (δH 5.74 (dd, J = 2.2, 9.2 Hz, H-13)/δC 137.3, δH 5.97 (dd, J = 2.8, 9.2, H-14)/δC 131.0, δC 133.4 (C-6), and 133.8 (C-15)) in the 1D NMR spectra. The two latter signals were attributed to a tetrasubstituted olefin which was unique for compound 4, along with only one CH3 group appearing as a doublet at δH 0.98 (d, J = 6.2 Hz, H-18)/δC 23.4. The second CH3 group observed as a doublet in compounds 1–3 appeared in the 1H NMR spectrum of 4 as a singlet (δH 1.20, H-17) and showed a HMBC correlation with the quaternary carbon at δC 70.1 (C-10) (Figure 4). As in compounds 1–3, resonances of a (2E,4E)-penta-2,4-dienoic acid moiety as well as three additional CH groups, two CH2 groups and one CH3 for the completion of the decalin part were observed.

The relative configuration of the stereogenic centers was determined by NOESY (Figure 4). Correlations were observed between H-7 (δH 1.56) and H-9ax (δH 1.06), H-11ax (δH 1.33), and the C-18 methyl protons (δH 0.98). An additional correlation was found between H-11ax (δH 1.33) and the C-17 methyl protons (H-17, δH 1.20) suggesting an equatorial orientation of the latter. From the complete spectral assignment, the structure of compound 4 was established as shown in Figure 5. The fourth member of the series was consequently named tanzawaic acid L (Figure 5).

Along with compounds 1–4, four known polyketides were isolated and identified (Figure 5) as arohynapene A (5) [3], arohynapene B (6) [3], tanzawaic acid A (7) [1], and tanzawaic acid E (8) [2]. Their structures were established based on NMR data and by comparison with those reported in the literature. The structures of arohynapene A and tanzawaic acid E were furthermore supported by X-ray crystallography (Figure 6). In the latter case, the absolute configuration could be determined even though no heavy atom is present in the unit cell. The result confirms the synthetic work of Arimoto et al. who determined the configuration of tanzawaic acid A to be 8R,10S[4] and suggests the other tanzawaic acids to have the same absolute configuration.

Tanzawaic acids were found in different Penicillium species, especially tanzawaic acid A which seems to be omnipresent in Penicillium citrinum[1,2]. Tanzawaic acids E and F were isolated from a marine-derived Penicillium steckii and were detected in at least 16 culture collection strains of the same species in a secondary metabolite study [2].

Recently, El-Neketi et al. [5] described the isolation of two new tanzawaic acids G and H, as well as tanzawaic acids B and F and arohynapene D from an endophytic Penicillium citrinum species. Therefore, Penicillium sp. IBWF104-06 appears to be related by its secondary metabolites to the aforementioned strains which do belong to the Penicillium section Citrina[6]. Tanzawaic acid E differs from tanzawaic acid L by two more hydrogen atoms at C-6 and C-15 and has the same elemental composition as tanzawaic acid K, the structural difference being the position of the OH group located at C-15 of tanzawaic acid K instead of C-10 in tanzawaic acid E [2]. Tanzawaic acids I and J have an 18 amu lower mass than hynapene A [7]. In contrast to the latter compound, both tanzawaic acids contain a double bond at C-14/15 instead of an OH group at C-14.

Biological activity. The isolated compounds were tested for their bioactivity in a series of antimicrobial and antiproliferative assays. We found that apart from compounds 1 and 2, the other isolates (3–8) inhibited the conidial germination in Magnaporthe oryzae at concentrations of 50 µg/mL or less (Table 3) whereas the germination of the grey mold Botrytis cinerea and the potato blight caused by the oomycete Phytophthora infestans were not affected at comparable concentrations. Even though several biological activities have been found for the known compounds 5–8, an inhibition of fungal spore germination of Magnaporthe oryzae was not reported to date. The structurally related hynapenes B and C showed antimicrobial activity against Pyricularia oryzae[8] whereas arohynapenes A, B, and D [3,9] were not active in agar diffusion assays. However, arohynapenes A and B inhibited the spore germination at 25 µg/mL (IC90). In addition, tanzawaic acid A (7) showed weak activity in agar diffusion assays at 50 µg/filter dics against Bacillus brevis, Mucor miehei, and Paecilomyces variotii, as well as cytotoxic effects against HeLaS3 cells at a concentration of 50 µg/mL. However, none of the other compounds showed bioactivity in these assays up to 50 µg/mL.

Table 3:
Inhibition of germination in Magnaporthe oryzae (IC90 represents the concentration at which 90% inhibition of conidial germination was observed).

Compound

M. oryzae IC90 [µg/mL]

tanzawaic acid I (1)

–

tanzawaic acid J (2)

–

tanzawaic acid K (3)

25

tanzawaic acid L (4)

50

arohynapene A (5)

25

arohynapene B (6)

25

tanzawaic acid A (7)

10

tanzawaic acid E (8)

25

Experimental

General experimental procedures. Optical rotations were measured on a Perkin Elmer polarimeter model 241 at 546 and 578 nm and were extrapolated to 589 nm using Drude’s equation [10]. 1H, 13C and 2D NMR spectra were recorded on a Bruker AVANCE III 600 MHz spectrometer equipped with a 5 mm TCI cryogenic inverse probe head (z-gradient) using standard pulse sequences. APCI-MS spectra were measured from a solution of the analyte in MeCN/H2O with a Hewlett Packard MSD 1100 using an evaporator temperature of 400 °C and a drying gas temperature of 350 °C at a flow of 6 L/h (N2). In positive ionization mode, the capillary voltage amounted to 3.5 kV, the corona discharge current was 4 µA. In negative ionization mode, the capillary voltage amounted to 2.2 kV, the corona discharge current was 6 µA. HRMS-ESI data were recorded on a Q-ToF ULTIMA-III (Waters) equipped with a LockSpray-interface. A Bruker IFS48 FTIR spectrometer and a Perkin-Elmer Lambda-16 spectrophotometer were used to measure the IR and UV spectra, respectively.

Producing organism. The producing fungus IBWF104-06 was isolated from a soil sample. The strain was determined as a Penicillium species by morphology. Strain IBWF104-06 has been deposited in the culture collection of the Institute of Biotechnology and Drug Research (IBWF e.V.), Kaiserslautern, Germany. For maintenance, the fungus was grown on agar slants on YMG agar (yeast extract 4.0 g/L, malt extract 10 g/L, glucose 10 g/L, the pH value was adjusted to 5.5 before sterilisation). Solid media contained 2.0% agar.

Bioactivity assays. Antimicrobial activities against bacteria and fungi were determined in the agar plate diffusion assay as described previously [12]. Cytotoxicity was assayed as described previously [13]. The cell line HelaS3 was grown in DMEM medium (Invitrogen). The medium was supplemented with 10% heat-inactivated fetal calf serum (Invitrogen), 65 mg/L of penicillin G and 100 mg/L of streptomycin sulfate. The viability was evaluated by microscopy after 72 h. The spore germination was tested with M. oryzae as described previously [14]. This method was adapted for the spore germination assay with Phytophthora infestans and Botrytis cinerea.